Defects in DNA transcription lead to genetic instability and mis-regulation of genetic programs; these characterize many cancers and developmental defects. In eukaryotic cells mRNA transcripts are produced from nucleosomal templates, where the DNA is tightly wrapped around cores of histone octamers. Nucleosomes are formidable obstacles to proteins that must access DNA. A wide variety of chromatin modifying enzymes regulate this barrier by adding or removing a large variety of histone modifications to nucleosomes. However, the compositional stability of nucleosomes in transcribed regions has largely been assumed. Our recent work suggests that this assumption is incorrect. While bulk histones are stable after they are deposited during DNA replication, we showed that the highly conserved histone variant H3.3 targets actively transcribed regions and deposits by a distinct, replication-independent (RI) chromatin assembly pathway. The partner histone H4 also undergoes RI deposition, indicating that entire nucleosomes are disassembled and replaced. Our results imply that transcriptional activity damages chromatin by disrupting nucleosomes. The function of RI chromatin assembly is unknown, but we are considering three essential functions: H3.3 may be required 1) to rebuild disrupted chromatin, 2) to add and remove histone modifications from active and repressed chromatin, or 3) to drive developmental transitions in chromatin structure. The long-term goal of this project is to test these hypotheses by analyzing in vivo functions of H3.3 and its partner H4 in Drosophila melanogaster cultured cells and genetic strains. The H3.3 histones in Drosophila and in humans are 100% identical, so this project is of broad relevance. Mutation of one mammalian H3.3 gene is lethal, arguing that RI deposition is an essential process. The specific aims include: 1) defining the timing and sites of H3.3 deposition to gain insight into the mechanism of RI chromatin assembly, 2) measuring the rates of histone replacement to define effects on chromatin stability, 3) characterizing the components and properties of the H3.3-containing nucleosome, 4) analyzing the effects of specified mutations in replacement histone genes to define their essential functions, and 5) determining the roles of proteins included in a H3.3-pre-deposition complex in RI chromatin assembly and transcription from chromatin templates. H3.3 becomes greatly enriched in the chromatin of neurons and germline cells, and it is therefore critical to determine if defects in RI chromatin assembly lead to neural dysfunction and infertility. [unreadable] [unreadable]